Alcohol drinking and certain pathophysiological conditions such as fasting and diabetes increase the levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and other P450 enzymes in humans and animal models. It is now known that CYP2E1 can metabolize more than 70 substrates of different chemical structures. These CYP2E1 substrates include: ethanol, acetaldehyde, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, long chain fatty acid including arachidonic acid, and nitrosamines. Increased CYP2E1 leads to elevated production of acetaldehyde, reactive oxygen species, free radical metabolites and lipid peroxides while reducing cellular anti-oxidants such as glutathione. Therefore, cells or tissues with increased CYP2E1 become more susceptible to damage or cell death, especially in the presence of an additional challenge. In the past, we have cloned the genes for human and rat CYP2E1 and demonstrated multiple regulatory mechanisms. Although other scientists already demonstrated cell damage (apoptosis and necrosis) by alcohol and acetaminophen using in vitro models, the molecular signaling mechanisms for their toxicities were not shown. During the last two or three years, we have been studying the role of elevated CYP2E1 in cell damage caused by alcohol and other CYP2E1 substrates and their mechanisms. Unlike other investigators who used CYP2E1-transfected HepG2 cells, we used C6 glioma cells since these cells contain CYP2E1, albeit in a small quantity, and undergo apoptosis upon exposure to CYP2E1 substrates. Therefore, we investigated our initial hypotheses that CYP2E1 substrates and their metabolites would activate the c-Jun N-terminal protein kinase (JNK) and p38 mitogen activated protein (MAP) kinase associated with cell death pathway while they would suppress the enzymes involved in the cell survival pathway. In addition, inhibition of CYP2E1 and JNK elevated during apoptosis effectively prevents cell death caused by CYP2E1 substrates. Our results showed that APAP caused time and dose-dependent apoptosis of C6 glioma cells. Activities of JNK and its immediate upstream kinase SEK-1 were rapidly increased 15 min after APAP exposure and this effect lasted up to 4 h. However, APAP did not activate the following enzymes: p38 MAP kinase, extracellular-signal regulated protein kinase (ERK), phosphatidylinositol 3-kinase, and Akt protein serine/threonine kinase. APAP-induced cell death was preceded by elevation of cytochrome c release and activation of caspase 3, a critical enzyme in executing apoptosis. We then demonstrated the critical role of the selective JNK activation in APAP-induced apoptosis by three key experiments: 1) transient transfection of the cDNA for JNK wild type or the dominant negative JNK KR mutant followed by cell death rate measurement; 2) differential effects of cytotoxic APAP and its non-toxic analog 3-hydroxyacetanilide on JNK activation and cell death rate; and 3) efficient blockade of JNK activation and cell death by pretreatment of C6 cells with the CYP2E1 inhibitor, YH439, which effectively suppressed the levels of CYP2E1 activity and protein content. These data indicate the critical role of CYP2E1-dependent metabolism and JNK activation during apoptosis. Our result of the selective JNK activation, therefore, is in contrast with other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor 1 alpha and interleukin 1 beta, all of which activate p38 MAP kinase along with the JNK in a coordinated fashion. Our in vitro results were also observed in in vivo models where APAP and carbon tetrachloride selectively and transiently increased the activities of JNK and SEK-1. Because of the critical role of selective activation of JNK-related pathway in cell damage caused by the three CYP2E1 substrates (APAP, 4-hydroxynonenal, and carbon tetrachloride), we are investigating whether other CYP2E1 substrates (including ethanol and arachidonic acid) cause cell damage by a similar mechanism.